The dinoflagellates are important microscopic members of the planktonic community. There are believed to be over 2000 living species, including those of the genus Gymnodinium, Karenia, Prorocentrum, Alexandrium, Symbiodinium, Crypthecodinium, Noctiluca, Gonyaulax, Protoperidinium, Gyrodinium, Amphidinium and Scrippsiella. Primarily, though not exclusively, they are marine plankton. Non-photosynthetic dinoflagellates feed on diatoms or other protists. Many are photosynthetic and are important primary producers in coastal waters. Some are symbiotic, living in the cells of their hosts, such as corals and sea anemones.
Dinoflagellates are of importance for several reasons. First, they are known to produce a wide spectrum of bioactive natural products, including neurotoxins, some of which can act on humans (e.g. paralytic shellfish poisoning, the worst cases of which result in respiratory failure and death within 12 hours, ciguatera poisoning and diarrhetic shellfish poisoning). Some of these toxins are channel modulators (e.g. saxitoxins and maitotoxins) that are currently used in research on areas such as ion channel mechanisms. Furthermore, many have potentially useful pharmacological activity, e.g. the carbenolides and amphidinolides which show very promising anti-tumor properties.
Second, some dinoflagellates (e.g. species of the genus Crypthecodinium) produce large quantities of omega-3 fatty acids, particularly docosahexaenoic acid (DHA). These polyunsaturated fatty acids are known to be beneficial in reducing the incidence of coronary heart disease and are therefore included in a variety of health products. DHA is also used in infant formulas due to its high incidence in human milk and its implication in brain development.
Third, dinoflagellates are mainly responsible for the so-called red tides that occur in many seas worldwide. These red tides are caused by a massive multiplication (or “bloom”) of dinoflagellates, usually in warm saltwater. The precise cause of these red tides is not known, although some experts believe that high temperatures combined with a lack of wind and rainfall are usually the catalysts for these blooms. These red tides can have major economic and health implications. The large quantities of toxins that are produced by the dinoflagellates in these red tides can not only kill a large range of marine species but they can also be accumulated in high concentrations in shellfish which are immune to the toxins and can result in severe digestive complaints, respiratory problems and even death in humans that eat these shellfish.
Because of their importance, it is desirable to produce live pure isolates and cultures of dinoflagellates, both to study them (to better understand red tides, for example), and to cultivate them to screen for bioactive natural products and to produce large quantities of high purity omega-3 fatty acids such as DHA. Unfortunately, until now it has been very difficult to generate such live pure isolates. This is due to the slow growth rate of dinoflagellates relative to other unwanted species of phytoplankton and to the inability of many dinoflagellates to grow in artificial media. The conventional method involves the isolation of individual cell(s) of the desired dinoflagellate from environmental samples. The pre-cultures are then incubated, either in seawater or other nutrient enrichments. However, as seawater and nutrient enrichments are not selective to dinoflagellates, other groups of faster-growing contaminating phytoplankton usually dominate or completely overtake the pre-cultures. Using such standard techniques, it typically takes a minimum of six months to scale up from a single-cell isolate to a 10 litre culture of the desired dinoflagellate. The success rate of making cultures from a single-cell isolate is typically of the order of 20%. Clearly, an improved method for the production of live pure isolates and cultures of dinoflagellates is highly desirable.
Leucaena leucocephala is a tropical and subtropical legume widely used in agroforestry systems throughout the world. It has been hailed as the perfect tree as it can serve many purposes, as foliage for livestock, as fuel wood or as green manure (1). Introduction of Leucaena outside its indigenous range has often led to acute and chronic toxicosis in animals (14). The agents of toxicity are the allelochemicals mimosine (α-amino-3-hydroxy-4-oxo-1-pyridine propanoic acid), a non-protein amino acid, and its main degradative product 3,4-dihydroxypyridine (DHP) (2). The concentrations of mimosine in air-dried Leucaena leaves were found to be in the range of 2.5-5.75% (2) and can be easily removed by soaking in water for 24 h (2). Soil extracts from Leucaena plantation also have phytotoxicity to other plants (12). Mimosine and toxic degradative products thereof such as DHP are known to be toxic to all eukaryotic cells and most bacteria.